Pharmacokinetics and bioequivalence evaluation of omeprazole and sodium bicarbonate dry suspensions in healthy Chinese volunteers

Omeprazole and sodium bicarbonate dry suspension are effective treatments for acid-related disorders. This study compared the bioequivalence and safety of the two formulations of omeprazole and sodium bicarbonate powder and assessed how CYP2C19 gene polymorphisms affect pharmacokinetics (PK). A single-center, randomized, single-dose, 2-sequence and 2-period crossover method was performed in forty healthy Chinese subjects. Blood samples were collected after a single dose for PK (AUC0–∞, AUC0–t, and Cmax) analysis. The concentrations of Omeprazole in human plasma were determined by HPLC–MS/MS. Besides, the gene polymorphisms of CYP2C19 were assessed by Sanger sequencing. The geometric mean ratios (90% confidence interval) [GMR (95% CI)] of Test/Reference preparation for Cmax: 95.2% (88.48%, 102.43%), AUC0–t: 97.47% (94.4%, 101.02%), AUC0–∞: 97.68% (94.27%, 101.21%) were within the range of 80.00–125.00%. The non-parametric test showed no statistical difference in Tmax between the two groups (p > 0.05). All drugs were well tolerated, no severe adverse reactions occurred, and no significant differences in adverse events between the two drugs. For CYP2C19 gene polymorphisms, the results showed that of 40 subjects, 12 subjects were extensive metabolizers, 24 were intermediate metabolizers, and 4 were poor metabolizers, the frequency of metabolic genotypes were 30%, 60%, and 10%. And the allele distributions for CYP2C19 were *1, *2, and *3 at 60%, 38.75%, and 1.25%. Both the CYP2C19 alleles and metabolic genotypes were consistent with other studies in Chinese. The results of PK parameters showed that different genotypes of CYP2C19 lead to significant differences in t1/2, AUC0–t, AUC0–∞ and Cmax, but no significant differences in Tmax in each group. At the same time, we confirmed that the PK parameters of the test and reference had no differences between the males and females. This study has shown that the pharmacokinetic parameters of the two formulations are not significantly different, which showed bioequivalence and exemplary safety. CYP2C19 gene polymorphism significantly differed in the PK parameters of omeprazole sodium bicarbonate powder.

Study design. This study was a single-dose and two-period PK study, shown in Fig. 1.
Forty subjects were randomly divided into two groups, with 20 patients in each group. The drugs were given to the patients of each group in the order of T-R and R-T in two cycles, with a single dose of 1 bag (each bag: 20 mg omeprazole + 1680 mg sodium bicarbonate). The subjects in each group fasted after 21:00 the night before administration. Collecting plasma samples at 0 h (within 60 min before administration) and after administration of 5 min, 10 min, 15 min, 20 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 8 h, 10 h, 12 h (a total of 16 points) and stored at − 80 °C until analysis. The blood samples were centrifuged at 1700 g (4 °C) for 10 min within 1 h of collection. The centrifuged plasma was immediately aliquoted into 2 tubes with corresponding labels (1 for testing, 1 for backup, the volume of plasma in each tube should not be less than 500 µL) and stored at ATPase in the activated form 80 °C until analysis. The total time from whole blood collection to centrifugation to separate plasma and then stored in ATPase in the activated form 80 °C refrigerator should not exceed 2 h. Analytic methods. The concentration of omeprazole in EDTA-K2 anticoagulant human plasma was determined by HPLC-MS/MS, quantified by internal standard (internal standard: omeprazole-D3). The sample pretreatment method was the protein precipitation method. And the linear range of the omeprazole plasma concentration determination method was 4-4000 ng mL −1 , the minimum quantitative limit was 4 ng mL −1 .
Pharmacokinetics analysis. The pharmacokinetic (PK) parameters evaluated in this study included maximum plasma omeprazole concentration (C max ) and time to reach the maximum plasma concentration (T max ) obtained directly from non-interpolated data, as well as the area under the plasma concentration curve of omeprazole at 0-t after administration (AUC 0-t ) using the linear trapezoidal method to calculate. Terminal elimination rate constant λz and the apparent terminal elimination half-life (T 1/2 ) were also needed. AUC 0-∞ (the AUC from time 0 to infinity) used the formula: AUC 0-∞ = AUC 0-t + Ct/λ(T 1/2 = 0.693/λz) to calculate. Statistical methods. SAS 9.4 software was used for statistical analysis. After logarithmic conversion, C max , AUC 0-t, and AUC 0-∞ performed a two-way unilateral t-test to calculate the 90% confidence interval of the geometric mean ratio of omeprazole C max , AUC 0-t, and AUC 0-∞ in the plasma of tested preparation T and reference preparation R. When the 90% confidence interval of the geometric mean ratio of C max , AUC 0-t, and AUC 0-∞ between the tested and the reference preparation was within the equivalent interval of 80.00-125.00%, the bioequivalence of the two preparations could be determined. Besides, the nonparametric method was used to evaluate the T max of test and reference preparation.

Effects of CYP2C19 phenotypes on PKs.
Of the 40 subjects completing the study, 40 subjects were divided into EM (CYP*1/*1, N = 12), IM (CYP*1/*2, N = 23, CYP*1/*3, N = 1), and PM (CYP*2/*2, N = 4). The AUC 0-t , AUC 0-∞ , C max , T 1/2 , λz, CL, and V d of test and reference preparation in each metabolic genotype were shown as mean ± SD in Table 3. According to the CYP2C19 phenotype, the mean plasma concentra-   www.nature.com/scientificreports/ tion-time profiles of the test preparation and the reference preparation were shown in Fig. 3. The plasma concentration of test and reference preparation in PM were much higher than that in EM and IM. In the PM of the test preparation, the C max was 1650.00 ± 451.66 ng mL −1 , which was significantly higher than that in EM (633.25 ± 295.94 ng mL −1 , p < 0.01) and IM (1044.21 ± 320.07 ng mL −1 , p < 0.001). And in the PM, the AUC 0-t was 4526.56 ± 651.59 ng h mL −1 , which was significantly higher compared with EM (552.90 ± 391.42 ng h mL −1 , p < 0.001) and IM (1520.14 ± 1437.90 ng h mL −1 , p < 0.001). Likewise, T 1/2 was also the highest in PM compared with the other groups (p < 0.001). In contrast, the drug clearance of the PM was the lowest among the three groups (p < 0.001). However, in terms of absorption, there was no significant difference in the T max in the three groups of the test preparation in vivo (p > 0.05). Similarly, significant differences were also found in C max , AUC 0-t , AUC 0-∞, T 1/2 , and λz of the reference preparation between different CYP2C19 genotypes. In contrast, there were no differences in the T max of reference in three groups, p > 0.05. In general, no matter whether it was the test or the Table 3. The PK of test and reference preparation about CYP2C19 phenotypes. C max maximum blood concentration, T max time to maximum blood concentration, AUC 0-t Area under curve from time 0 (baseline) to time t, AUC 0-∞ Area under curve from zero to infinity, T 1/2 elimination half-life, λz apparent end elimination rate constant, CL plasma clearance, V d , apparent volume of distribution. Data was presented in mean ± standard deviation. p > 0.05, not significant.  www.nature.com/scientificreports/ reference preparation, the C max , AUC 0-t , and AUC 0-∞ in the EM, IM, and PM groups were gradually increased (p < 0.05), and T 1/2 and λz were gradually decreased (p < 0.05), except that there was no difference in T 1/2 and λz between the EM and IM groups in the reference preparation (Fig. 4). For that, the CYP2C19 phenotypes have little effect on the absorption of drugs in the human body, and the main effect lies in drug metabolism.

Discussion
Omeprazole has been widely recognized and used as the first generation of new acid inhibitors once discovered. Different enteric coatings are necessary to protect acid unstable PPI from gastric acid degradation within the stomach, which has the potential detriment of PPI absorption delayed 18 . But omeprazole sodium bicarbonate dry suspension can overcome this problem. Sodium bicarbonate can not only protect omeprazole from being destroyed by gastric acid 23 , but also can quickly neutralize gastric acid, increase the pH value in the stomach, www.nature.com/scientificreports/ relieve some clinical symptoms, and activate the proton pump channel in a large amount. Omeprazole can directly act on the proton pump channel to inhibit the secretion of gastric acid by the proton pump. The first purpose of this study is to find out the bioequivalence of the test and reference preparation. After the single administration, the exposure of the test and reference preparation was similar. The GMR (90%CI) for C max , AUC 0-t , and AUC 0-∞ were all between 80 and 125%. In addition, both preparations were well tolerated without any serious adverse events. There were no newly reported adverse events in the present study, and there was no significant difference in the frequency of drug-related adverse events between these two formulations.
Omeprazole has highly variable pharmacokinetics, of which CYP2C19 is a major influencing factor 24,25 . The CYP2C19 gene is extensively polymorphic with 39 known alleles 26 , belonging to an important drug-metabolizing enzyme in the liver cytochrome P450 enzyme series. The frequency alleles of CYP2C19 tend to differ in relation to race 27,28 . The CYP2C19*2 and CYP2C19*3 are responsible for PM alleles, mainly found in Asians. The CYP2C19*2 has an allele frequency of 25-30% in Asians and about 15% in whites 29 , and CYP2C19*3 has an allele frequency of about 2-7% in Asians 29 while 0.04% in whites 30 . Of the 40 volunteers in the present study, the allele distributions for CYP2C19 were *1, *2, and *3 at 60%, 38.75%, and 1.25% close to the ratio in the Asian described above. Besides, it was similar to the allele distributions for CYP2C19 in other Chinese studies, that the *1, *2, and *3 were 58.2-69.7%, 24.7-37.7%, and 2-4.1% [31][32][33][34] . While the frequency of the CYP2C19 EM, IM, and PM genotypes in the present study were 30%, 60, and 10%, which is similar to other CYP2C19 gene polymorphisms performed in Chinese, the proportions of EM, IM, and PM were 27.5%, 57.5%, and 15%, respectively 35 . The results in the present study showed that CYP2C19 was crucial for omeprazole pharmacokinetics in vivo. The CL was significantly higher in EM, compared with it in PM and IM. The most representative PK parameters-AUC, reflecting the drug clearance, which was significantly increased in PM 8.2, 3 times in EM and IM, consistent with previous studies 29 . The other PK parameters C max and T 1/2 were also significantly different according to different genotypes (Fig. 4). Alterations in PK parameters of omeprazole were found in several studies in different races [36][37][38][39][40] . In the EM group of the present study, the AUC 0-t of omeprazole was 552.9 ± 391.42 ng h mL −1 , which was higher than 250.5 ± 16.1 ng h mL −1 in West Asian 37 , but not different from the reports in Whites (635.5 ± 259.7 ng h mL −1 ) 36 , and East Asians (618.3 ± 141.9 ng h mL −1 in Japanese 38 and 713.49 ± 555.56 ng h mL −1 in Korean 39 ). This result showed that Caucasian has the least growth of omeprazole AUC 0-t between the EM group and PM group than those in East and West Asians. In the EM group, the omeprazole AUC 0-t of Caucasian was comparable those of East Asians, but still higher than that of West Asians. In the PM group, the omeprazole AUC 0-t Caucasian was significantly lower than that of East Asians, but still higher than that of West Asians. Our data is closer to the reported pharmacokinetic data in Chinese 40 and Japanese population. In PM group, the omeprazole AUC 0-t value is significantly higher than that of Caucasians and West Asians. At the same time, the increment of omeprazole AUC 0-t from EM to PM groups is close to 8 times, which is much higher than 5 times enhance in those of Caucasians and Koreans, and lower than 9 times in West Asians. Omeprazole total exposure differs among different races for the same metabolic phenotype, specifically in the PM. There in the PM, the C max was 635.5 ± 259.7 ng mL −1 in Caucasians and 538.6 ± 33.5 ng mL −1 in East Asians, lower than 1070.2 ± 185.3 ng mL −1 in Japanese and 1650.00 ± 451.66 ng mL −1 in present study. In the EM group, the C max of this study was 633.25 ± 295.94 ng mL −1 , higher than that 285 ± 82.9 ng mL −1 in Caucasians and 251.1 ± 46.2 ng mL −1 in Japanese, and 152.4 ± 9.94 ng mL −1 in West Asians was lowest. These showed that the absorption of omeprazole in EM and PM of this study were higher than those in Caucasian, West Asians and Japanese. In the EM, the C max in the Caucasians was similar with that of Japanese, but higher than that in West Asians. But in the PM, the C max in the Caucasians was close to that in West Asians, and lower than that in Japanese. Besides, the data showed that the increment of omeprazole C max from EM to PM in this study is close 3 times, which is lower than 4 times enhance in those of West Asians and Japanese, and higher than 2 times in Caucasians. For the half-time of the omeprazole in these populations, in the EM group, the T 1/2 in this study was 0.74 ± 0.19 h, close to 0.71 ± 0.1 h in Caucasians, but lower than 0.9 ± 0.01 h in West Asians and 1.09 ± 0.08 h in Japanese. And in the PM, the T 1/2 was 2.48 ± 0.24 h, close to 2.68 ± 0.3 h in Caucasians, 2.42 ± 0.18 h in West Asians and 2.41 ± 0.15 h in Japanese, which indicated that the half-time of omeprazole in the PM of these populations are similar.
In addition, gender may also affect the pharmacokinetic parameters of drugs by affecting the activity of CYP2C19, thus changing the drug's efficacy. Shabnam Nazir's study showed that the C max and AUC 0-t of Table 4. The PK of test and reference preparation in males (N = 24) and females (N = 16) healthy volunteers. C max maximum blood concentration, T max time to maximum blood concentration, AUC 0-t AUC from time 0 (baseline) to time t, AUC 0-∞ AUC from zero to infinity, T 1/2 elimination half-life, λz apparent end elimination rate constant. Data was presented in mean ± standard deviation. p > 0.05, not significant. www.nature.com/scientificreports/ omeprazole in females (2.913 ± 0.61 μg mL −1 and 8.74 ± 2.23 μg h mL −1 ) was significantly higher than those (2.006 ± 0.98 μg mL −1 and 6.67 ± 4.32 μg h mL −1 ) in males. At the same time, the C max and C max of 5-hydroxyomeprazole and omeprazole siphon of women were greatly higher than those of men, which meant that there were significant differences in CYP2C19 activity between females and males in Pakistani 41 . In another study in the Korean population, for the same CYP2C19 genotype, Korean women metabolized omeprazole faster than Korean men 42 . However, An Iranian study reported that there was no difference in the hydroxylation index of omeprazole between females and males 43 . And this study showed that the PK parameters of the test and reference preparation had no differences between males and females, which was consistent with the other studies in Chinese 44 and Whites 45 . Taken together, the sex dependence of CYP2C19 activity may be related to the race included in the study.
There are several limitations in this study. First, only healthy volunteers were enrolled in the present study, the omeprazole concentration-time profile of which may differ from the acid-related disordered patients. Secondly, the number of enrolled volunteers should be expanded to meet the needs of volunteers for pharmacokinetic research in PM. Thirdly, the efficacy of omeprazole was not evaluated, which exerted its drug effect by inhibiting gastric acid secretion. This study failed to evaluate the intragastric 24 h pH or serum gastrin for its efficacy.

Conclusion
The results showed that the GMR (90% CI) Cmax, AUC 0-t , and AUC 0-∞ were all between 80 and 125%, which meant that the test and reference preparation are bioequivalent. And there were no serious AEs that occurred during the trials, indicating that both medications are well tolerated and have exemplary safety in healthy Chinese volunteers. Besides, our study demonstrated that CYP2C19 gene polymorphism significantly differed in the PK parameters of omeprazole sodium bicarbonate dry suspension.

Data availability
The datasets used and analyzed during the current study available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.